TITLE: Examination of the Role of DNA Methylation Changes in Prostate Cancer using the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model

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1 AD Award Number: W81XWH TITLE: Examination of the Role of DNA Methylation Changes in Prostate Cancer using the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model PRINCIPAL INVESTIGATOR: Shannon R. Morey CONTRACTING ORGANIZATION: Roswell Park Cancer Institute Buffalo, NY REPORT DATE: March 2008 TYPE OF REPORT: Annual Summary PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland DISTRIBUTION STATEMENT: Approved for Public Release; Distribution Unlimited The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision unless so designated by other documentation.

2 REPORT DOCUMENTATION PAGE Form Approved OMB No Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports ( ), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. 1. REPORT DATE (DD-MM-YYYY) 2. REPORT TYPE Annual Summary 3. DATES COVERED (From - To) 1 MAR FEB TITLE AND SUBTITLE 5a. CONTRACT NUMBER Examination of the Role of DNA Methylation Changes in Prostate Cancer using the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model 6. AUTHOR(S) Shannon R. Morey Shannon.MoreyKinney@RoswellPark.org 5b. GRANT NUMBER W81XWH c. PROGRAM ELEMENT NUMBER 5d. PROJECT NUMBER 5e. TASK NUMBER 5f. WORK UNIT NUMBER 7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8. PERFORMING ORGANIZATION REPORT NUMBER Roswell Park Cancer Institute Buffalo, NY SPONSORING / MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSOR/MONITOR S ACRONYM(S) U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland SPONSOR/MONITOR S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT Approved for Public Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT The TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) model provides an excellent system to study disruption of the DNA methylation process in prostate cancer. To date, several key conclusions can be made from this research. First, analysis of methylation patterns in TRAMP revealed a small number of hypermethylation events in early stage lesions, with a great increase in late stage tumors. Furthermore, late stage tumors, androgen independent tumors and metastases each display numerous and tumor type specific hypermethylation events. Secondly, a large proportion of these hypermethylated genes, including p19/arf and p16ink4a, display downstream hypermethylation correlating with robust overexpression. In addition, p16 and p19 overexpression, but not downstream hypermethylation, occurs in early stage prostatic lesions in TRAMP, suggesting that overexpression may be the initiating event and pharmacological reversal of downstream hypermethylation in TRAMP cell lines led to decreased expression of p19 and p16, indicating that downstream hypermethylation contributes to the maintenance of increased gene expression. Overall these data indicate that locus specific hypermethylation is selected for upon tumor progression and treatment with hypomethylating agents may inactivate oncogenes whose expression is maintained by downstream hypermethylation. 15. SUBJECT TERMS Prostate cancer, DNA methylation, Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) 16. SECURITY CLASSIFICATION OF: 17. LIMITATION OF ABSTRACT a. REPORT U b. ABSTRACT U 18. NUMBER OF PAGES c. THIS PAGE U UU 63 19a. NAME OF RESPONSIBLE PERSON USAMRMC 19b. TELEPHONE NUMBER (include area code) Standard Form 298 (Rev. 8-98) Prescribed by ANSI Std. Z39.18

3 Table of Contents Page Introduction Body.. 5 Key Research Accomplishments... 8 Reportable Outcomes 9 Conclusion 11 References 12 Appendices

4 Introduction: DNA hypermethylation of tumor suppressor gene promoters, in conjunction with hypomethylation of repetitive elements and increased expression of DNA methyltransferases (DNMTs), occurs in human prostate cancer. An understanding of how DNA methylation becomes deregulated in prostate cancer and how to reverse or prevent this process is important for developing anticancer therapies. It has also been shown that pharmacological inhibition of DNMTs can have anticancer effects, supporting the concept that hypomethylation and thus reexpression of tumor suppressor genes may have therapeutic significance in the treatment of cancer. The TRansgenic Adenocarcinoma of Mouse Prostate (TRAMP) SV40 transgenic autochthonous model, along with clonal cell lines derived from TRAMP primary tumors, provides an excellent system to study disruption of the DNA methylation process in prostate cancer and to determine whether inhibition of DNMTs abrogates prostate tumorigenesis. Our preliminary data suggest that DNA methylation is deregulated in the TRAMP model, which is characterized by altered methylation patterning of CpG islands and significantly increased DNMT activity and expression. Based on these findings, we hypothesize that aberrant DNA methylation contributes to TRAMP tumorigenesis, and that disruption of DNMTs will inhibit prostate oncogenesis in TRAMP. The information gained from this study will permit a better understanding of the role of aberrant DNA methylation in prostate cancer. Specific Aims: 1. Identify and characterize the biological significance of genes that have altered DNA methylation status in TRAMP. 2. Determine whether genetic disruption of DNMT1 inhibits prostate tumorigenesis in TRAMP. 4

5 Body: Examination of the Role of DNA Methylation Changes in Prostate Cancer using the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) Model Task 1. Identify and characterize the biological significance of genes that have altered DNA methylation status in TRAMP: To complete this task I analyzed several types of TRAMP samples including Prostatic Intraepithelial Neoplasia (PIN), Well Differentiated disease (WD), Early and Late Poorly Differentiated disease (EPD and LPD), Androgen Independent Primary (AIP) tumors and metastases from liver and lung (MET) (1-3). I then performed RLGS spot cloning to identify several loci that were commonly methylated in these different types of TRAMP tumors (Appended publications 1 and 2, (1, 3). While several of the identified genes were hypermethylated in the promoter region, a very high percentage of these genes showed hypermethylation of downstream regions rather than in the promoter of the gene (Table 1, 2, (1, 3)). I next performed qrt-pcr analysis to determine the expression of several of the commonly hypermethylated genes from the previous task. I examined those that displayed hypermethylation by RLGS analysis of either the promoter region or downstream regions. From these experiments I did not identify any genes that showed promoter region hypermethylation with correlating decreased expression. Therefore, the only gene fitting these criteria that I have identified in TRAMP is the IRX3 gene (Figure 1, (2)). However, I did identify six genes that display downstream hypermethylation correlating with increased expression, which we reported in two publications included in the appendix (1-3). These genes include p19, p16, Cacna1a, Gsc, Nrxn2, and the unknown gene BC (1-3). Perhaps the most interesting of these are p19 and p16 which are both encoded in the CDKN2A locus, since this locus was hypermethylated in almost all PD, AIP and Met tumors. I find that when these genes are overexpressd in LPD, AIP, or MET tumors they are hypermethylated in the third exon (Figure 2a-b, (1, 2)). I also find that these genes are overexpressed in PIN and WD samples that are not hypermethylated (Figure 2c-d, Table 1, (3)). These results suggest that the overexpression occurs prior to and may lead to the downstream hypermethylation. I also examined the expression of the six genes listed above in the C2D and C2N cell lines. The only genes that were expressed in these cell lines were p19 and p16. Furthermore when the cell lines were treated with a hypomethylating agent (5-aza-2 -deoxycytidine, DAC) the expression of both p19 and p16 was decreased (Figure 3a and data not shown). I also found that the CDKN2A locus is hypermethylated in TRAMP cell lines and this methylation is decreased when treated with DAC (Figure 3b). I then completed further methylation analyses on the downstream hypermethylated region identified by RLGS as well as the promoter region for p19, p16, Cacna1a, Gsc, Nrxn2, and the unknown gene BC (Figure 4 and appended publications (1, 3)). This was completed either through traditional bisulfite sequencing or Mass Array Quantitative Methylation Analysis (MAQMA) which is also based on the bisulfite conversion of DNA. However, no further methylation analysis was completed on genes identified by RLGS to have promoter hypermethylation since for none of these 5

6 genes was there any correlation with decreased expression. Bisulfite sequencing confirmed the RLGS results that the downstream regions were hypermethylated and that the promoters were unmethylated for each gene (Figure 4 and appended publications (1, 3)). I have not completed the later proposed experiments in this task (D and E) due to the unexpected results described above. However, I am currently performing methyl-dip chip array analyses on TRAMP tumors versus normal prostate to identify novel genes that display promoter hypermethylation correlating with decreased expression in TRAMP.I proposed this technique in the alternative approaches in the situation that RLGS analysis was not sufficient to identify such genes. The idea is that this more global approach where there are no limitations on gene identification will lead us to genes that fit the specific criteria. In addition to methyl-dip chip analyses I am currently examining several candidate tumor suppressor genes that are commonly hypermethylated in the promoter region associated with decreased expression, in human prostate cancer. These genes are Aldh1a2, Zfp185, Mgmt, Pdlim4, Rarres1, and Vegfr1. I hope to find that some of these genes are also hypermethylated in TRAMP to confirm that this phenomenon occurs in this model and as a set of genes that we can analyze in the Dnmt1 hypomorphic TRAMP mice to determine if DNA methylation changes that occur during TRAMP tumorigenesis are inhibited. Task 2. Determine whether genetic disruption of DNA methyltransferase 1 (DNMT1) inhibits prostate tumorigenesis in TRAMP: I did obtain C57Bl/6 mice carrying either the N or R Dnmt1 hypomorphic allele From Dr. Peter Laird and have produced sufficient mice in this breeding colony to complete the proposed experiments. The experimental design to characterize phenotype of DNMT1 hypomorphic mouse prostates and epigenetic parameters was to obtain a samples set of at least three mice for each possible genotype (WT, N/+, R/+, N/R) at either 15 or 24 weeks of age to analyze histologically for prostate development, for DNA hypomethylation at a global level, and for Dnmt protein expression. These experiments are nearly complete. However, the Dnmt1 hypomorphic alleles (N and R) are not inherited in a mendelian ratio (should be 1:1:1:1 for each genotype, WT, N/+, R/+, N/R), which was not previously reported. Mice having both the N and R alleles have the least amount of DNmt1 expression and therefore may show an altered phenotype compared to N/+ or R/+ which have only slightly less than normal Dnmt1 expression. Unfortunately N/R mice are found at much lower than expected (one fifth of expected) making these experiments take much longer than previously estimated (Table 3). I also analyzed animal weight, urogenital (UG) weight, and prostate weight in these animals and found that there is a significant decrease in all three parameters in N/R mice compared to WT mice at 15 weeks and in animal and UG weight at 24 weeks of age (Figure 5). The only other significant change observed was a decrease in UG weight in N/+ mice at 15 weeks. This may be explained by the observation that, overall there seems to be more difference at 15 weeks of age versus 24 weeks and that the N/+ mice have been shown to have less Dnmt1 expression than R/+ mice. Hematoxylin and eosin staining of prostate and liver in the Dnmt1 hypomorphic mice shows a normal morhology in WT, N/+, R/+ at 15 and 24 weeks of age and in N/R 6

7 mice at 15 weeks of age. I do not have samples for histology for N/R mice at 24 weeks of age due to the decreased Mendelian inheritance, but will obtain them shortly. Once the rest of N/R samples are collected I will perform immunohistochemical analysis for the proliferative marker Ki67 and other cell specific markers within the prostate to determine if the Dnmt1 hypomorphic prostates display a normal phenotype. In order to confirm that the genotype was correlated with the expected phenotype of DNA hypomethylation, I next measured global methylation (%5mdC/dG) in both prostate and liver tissues from the Dnmt1 hypomorphic mice at either 15 or 24 weeks of age. As expected, global hypomethylation is observed in N/R mice with slight hypomethylation in N/+ mice, with more hypomethylation at the earlier time point (Figure 7). I am currently completing western blot analyses of Dnmt1, Dnmt3a, and Dnmt3b proteins in samples from the four genotypes at either 15 or 24 weeks of age. The second part of this task is to produce 50:50 C57Bl/6 x FVB DNMT1 hypomorphic TRAMP mice. There are two breeding strategies for this. The first is a single cross of C57Bl/6 mice carrying either the N or R Dnmt1 hypomorphic allele to FVB TRAMP mice to produce 50:50 C57Bl/6 x FVB TRAMP mice carrying one DNMT1 hypomorphic allele. The second is to transfer the Dnmt1 hypomorphic alleles from the C57Bl/6 background to the TRAMP FVB background. These mice can then be backcrossed to C57Bl/6 mice carrying one Dnmt1 hypomorphic allele to produce 50:50 C57Bl/6 x FVB TRAMP mice carrying both DNMT1 hypomorphic alleles (N/R). I have collected tissue samples from several Dnmt1 hypomorphic TRAMP mice from the first breeding strategy at either 15 or 24 weeks of age. These mice can have one of three possible genotypes (WT, N/+, or R/+). The data collected at necropsy (animal weight, urogenital weight, prostate weight) show a statistically significant decrease in all three parameters in N/+ mice compared to WT mice at the 15 week timepoint, which is not seen at 24 weeks of age (Figure 8). The time it would take to produce the mice in the original second breeding strategy is in the order of years. Therefore, the number of backcrosses has been decreased from 7 to 4. The f4 mice are 93.75% FVB and offspring of a cross to C57 would be 46.9% FVB:53.1% C57. Because these mice are not purely 50:50 I will only use nonhypomorphic littermates as controls and will not collect any more of samples from the first breeding strategy. I am currently collecting samples at either 15 or 24 weeks of age from the 46.9% FVB:53.1% C57 mice which carry the TRAMP transgene and are of four possible Dnmt1 hypomorphic phenotypes (WT, N/+, R/+, N/R). Based on the finding that both Dnmt1 alleles are not inherited in mendelian fashion I will increase the number of breeding cages to obtain enough mice for this study. Once these samples are available I will begin characterizing the epigenetic parameters proposed for this task. 7

8 Key Research Accomplishments: Key Scientific Findings: Restriction Landmark Genomic Scanning (RLGS) analysis of methylation patterns in TRAMP revealed a small number of hypermethylation events in PIN and WD lesions, with a great increase in EPD and LPD tumors. LPD, AIP and MET tumor phenotypes each display numerous hypermethylation events, with the most homogeneous hypermethylation pattern in AIP tumors and the most heterogeneous hypermethylation pattern in metastases. There are several loci that displayed a tumor phenotype specific methylation status, suggesting that selection may play a role in the development of these patterns. Hypermethylated genes revealed by RLGS showed hypermethylation of downstream exons correlating with mrna overexpression. BC058385, Goosecoid (GSC), p19/arf, p16ink4a, NRXN2 and Cacna1a display downstream hypermethylation correlating with robust mrna overexpression. Overexpression of p16 and p19, but not downstream hypermethylation, occurs in early stage prostatic lesions in TRAMP, suggesting that gene overexpression is the initiating event. Pharmacological reversal of downstream gene hypermethylation in TRAMP cell lines led to decreased expression of p19 and p16, suggesting that downstream hypermethylation contributes to the maintenance of increased gene expression. N/R Dnmt1 Hypomorphic genotype is not inherited in Mendelian fashion N/R mice and less significantly N/+ mice are smaller in size and have decreased UG weight and prostate weight than WT mice N/R mice have significant hypomethylation in prostate and liver tissues compared to WT mice The differences between Dnmt1 hypomorphs and WT mice are more distinct at 15 weeks than 24 weeks of age Resources: Gene list of commonly hypermethylated loci in TRAMP from RLGS analysis Dnmt1 hypomorphic mouse colony (C57Bl/6) Dnmt1 hypomorphic TRAMP mouse colony (FVB) 8

9 Reportable Outcomes: Manuscripts Morey Kinney, Shannon R., Dominic J. Smiraglia, Smitha R. James, Michael T. Moser, Barbara A. Foster, and Adam R. Karpf. Stage-specific alterations of Dnmt expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the TRAMP model. Molecular Cancer Res. In press Marta Camoriano *, Shannon R. Morey Kinney*, Michael T. Moser, Barbara A. Foster, James L. Mohler, Donald L. Trump, Adam R. Karpf, and Dominic J. Smiraglia. Cancer Research. In press *Equal contribution Presentations Shannon R. Morey. Cancer Epigenetics as Seen Through the Eyes of a Mouse. Invited lecture 2007 Science Decade Lecture Series, Roswell Park Cancer Institute, March 6, 2007 Shannon R. Morey, Dominic J. Smiraglia, Barbara A. Foster, and Adam R. Karpf. Alterations in DNA Methylation During TRAMP Tumor Progression. Oral presentation at the Annual Pharmacology Sciences Day, University at Buffalo, May 14, Shannon R. Morey, Dominic J. Smiraglia, Michael T. Moser, Barbara A. Foster, and Adam R. Karpf. DNA Methylation Pathway Alterations in a Mouse Model of Prostate Cancer. Poster presentation at the AACR Edward A. Smuckler Memorial Pathobiology of Cancer Workshop, Snowmass, CO, July 18, Shannon R. Morey Kinney, Dominic J. Smiraglia, Michael T. Moser, Barbara A. Foster, and Adam R. Karpf. Comparison of Altered DNA Methylation During Prostate Cancer Progression Using the TRAMP Model. Poster presentation at the Pharmacology and Therapeutics Departmental Retreat. Holiday Valley Resort and Conference Center, Ellicottville, NY, November 8, Shannon R. Morey Kinney, Dominic J. Smiraglia, Michael T. Moser, Barbara A. Foster, and Adam R. Karpf. Comparison of Altered DNA Methylation During Prostate Cancer Progression Using the TRAMP Model. Poster presentation at the 14 th Annual Thanksgiving Poster Forum. Roswell Park Cancer Institute, Buffalo, NY, November 16,

10 Funding Shannon R. Morey Kinney, Marta Camoriano, Michael T. Moser, Barbara A. Foster, Dominic J. Smiraglia, and Adam R. Karpf. Restriction Landmark Genomic Scanning Reveals Phenotype Specific Epigenomic Patterns in a Mouse Model of Prostate Cancer. Poster presentation at the Keystone Cancer Genomics and Epigenomics Symposium, Taos, NM, February 21, SUM Mark Diamond Research Fund Award, University at Buffalo Epigenetic Deregulation in a Mouse Model of Prostate Cancer 10

11 Conclusion: While I was not able to identify any genes in addition to Irx3 that are hypermethylated in the promoter correlating with decreased expression in TRAMP, I did identify several genes that have increased expression that correlates with downstream hypermethylation. This phenomenon has been shown to occur in plants, but has not been well studied in mammals or cancer models. These data have been accepted for publication through peer review, indicating the importance of these unexpected findings. These results indicate that altered DNA methylation may play an important role in increased gene expression in addition to the well studied decreased gene expression. The overexpression of these genes may promote tumorigenesis and studies examining the oncogenic activity of these overexpressed genes in prostate cancer may lead us to identify novel therapeutic targets. Furthermore, these data suggest that treatment with hypomethylating agents may have dual activity of activating hypermethylated tumor suppressor genes as well as inactivating oncogenes whose expression is maintained by downstream hypermethylation. Future studies will be required to rigorously test this hypothesis. The previously unreported knowledge that the N/R Dnmt1 hypomorphic alleles are not inherited in Mendelian fashion as well as the knowledge that these mice seem to be runted to a certain extent will be very useful in completing the second task of this study. It is also important to report a complete characterization of this model. 11

12 References: 1. Camoriano, M., Shannon R. Morey Kinney, Michael T. Moser, Barbara A. Foster, James L. Mohler, Donald L. Trump, Adam R. Karpf, and Dominic J. Smiraglia. (2008). Phenotype-specific CpG Island Methylation Events in a Murine Model of Prostate Cancer. Cancer Research, In Press. 2. Morey, S. R., Smiraglia, D. J., James, S. R., Yu, J., Moser, M. T., Foster, B. A., and Karpf, A. R. (2006). DNA methylation pathway alterations in an autochthonous murine model of prostate cancer. Cancer Res, 66: Morey Kinney, S. R., Dominic J. Smiraglia, Smitha R. James, Michael T. Moser, Barbara A. Foster, and Adam R. Karpf. (2008). Stage-specific alterations of Dnmt expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the TRAMP model. Molecular Cancer Research, In Press. 12

13 Appendix Table 1. RLGS spot loss in TRAMP samples Number of times hypermethylated (%) Spot ID PIN n = 5 WD n = 6 EPD n = 7 LPD n = 14 Gene Name Gene Context of Hypermethylation Hypermethylation in CpG Island 3D22 0 (0) 0 (0) 7 (100) 14 (100) Cdkn2a 3' end no 4C13 0 (0) 0 (0) 5 (71) 13 (93) AK 'end yes 3D67 0 (0) 0 (0) 6 (86) 12 (86) Oprd1 3' end yes 3C21 0 (0) 0 (0) 6 (86) 10 (71) Nrxn2 5' end yes 4C31 0 (0) 0 (0) 7 (100) 10 (71) Adcy5 Body no 3E30 0 (0) 0 (0) 7 (100) 10 (71) Gsc 3' end yes 2G63 0 (0) 0 (0) 6 (86) 10 (71) BC Body yes 2C28 1 (20) 4 (67) 3 (43) 9 (64) AK 'end yes 5F09 0 (0) 0 (0) 3 (43) 9 (64) Cacna1a 3 end no 5B30 0 (0) 0 (0) 5 (71) 8 (57) ARHGEF17 5'end yes 2B37 0 (0) 0 (0) 1 (14) 7 (50) Ptprs Body yes 4C01 0 (0) 0 (0) 2 (29) 7 (50) AK 'end yes 6C17 0 (0) 0 (0) 1 (14) 6 (43) Foxd3 5' end yes 4D27 0 (0) 0 (0) 0 (0) 6 (43) Hoxa2 5' end yes 4C11 0 (0) 0 (0) 3 (43) 6 (43) N17Rik Body no 5D52 0 (0) 0 (0) 0 (0) 5 (36) Zar1 5' end yes 2D39 0 (0) 0 (0) 0 (0) 5 (36) CG 'end yes 4C17 0 (0) 0 (0) 3 (43) 5 (36) Lhfpl4 5'end yes 2D21 0 (0) 0 (0) 1 (14) 5 (36) BC 'end yes 4G73 0 (0) 0 (0) 2 (29) 5 (36) Irx3 5'end yes 4C38 0 (0) 0 (0) 1 (14) 4 (29) Lhfpl4 5'end yes 3D36 0 (0) 0 (0) 1 (14) 4 (29) Lmln 5' end yes 13

14 Table 1 RLGS spots of interest. PRIM MET AIP Total # Spot n=30 n=30 n=30 n=90 1 3C Class d P-value e CGI Context h Gene Homology a Prim or AIP 1.0E-12 Y Body Nrxn2 2 3E Prim or AIP 2.3E-11 N 3'end Gsc 3 5F Prim or AIP 2.5E-08 N Body Cacna1a 4 2G Prim or AIP 7.1E-08 Y Body BC B Prim or AIP 1.0E-05 f B Prim or AIP 1.2E-05 Y Body AK D Prim or AIP 7.0E-04 Y g 5'end Hoxa2 8 3E Prim or AIP 9.1E C Prim or AIP 3.0E-03 Y 5'end AK D Prim or AIP 3.0E G Prim or AIP 3.8E C Prim or AIP 7.0E C Prim or AIP 1.4E-02 Y 5' end Lhfpl4 14 4D b AIP specificity 2.0E-05 Y 5'end Nfyb 15 4E AIP specificity 5.0E-05 Y 5'end Tpm2 16 4D AIP specificity 6.0E-05 Y 5'end Pawr 17 4E AIP specificity 1.0E-04 Y 5'end Tpm2 18 3C AIP specificity 2.0E-04 Y 5'end Mid1ip1 19 3G AIP specificity 1.0E-03 Y 5'end Il6st 20 3D c Frequency N 3'end Cdkn2a 21 4C Frequency N Body N17Rik 22 4E Frequency C Frequency Y 5' end Lhfpl4 24 4C Frequency N Body Adcy5 25 4C Frequency Y 5'end AK D Frequency Y 3'end Oprd1 27 2C Frequency Y 5'end AK E Frequency D Frequency Y 3'end BC E Frequency Y 5' end Zfp E Frequency Y 5'end U2af1-rs1 32 6D Frequency N Intergenic Intergenic a Spots lost significantly in primary tumors plus androgen-independent primary tumors (n=60), but not metastatic tumors (n=30); b Spots lost significantly in androgen-independent primary tumors (n=30), but not in primary plus metastatic tumors (n=60); c Spots lost in greater than 30 samples from all tumor types (n=90); d Fischer s Exact test (two-tailed) for class membership; e Is the RLGS spot NotI site within 200bp of a CpG island; f RLGS spot is unidentified; g Within 5kb of transcriptional start site and/or including exon 1; h Annotated gene, mrna, or spliced EST within 5 kb of the CpG island or NotI site. 14

15 Figure 1. Promoter methylation of Iroquiox Homeobox Gene 3 (IRX3) is associated with decreased expression in TRAMP tumors. A) Upper: Diagram of IRX3 gene. Right arrow, transcriptional start site; open rectangles, exons; lines, introns; vertical arrow, position of the NotI site identified by RLGS; black bar, CpG island; horizontal line with circles, regions analyzed by sodium bisulfite sequencing.lower: Methylation analysis of NotI site identified to be hypermethylated by RLGS analysis and region upstream of transcriptional start site by bisulfite sequencing. Each circle indicates a CpG, with each row of circles indicating a sequenced clone. N Normal prostate, LPD - Late Poorly Differentiated. Filled circles indicate methylated CpGs and unfilled circles indicate unmethylated CpGs. B) qrt-pcr expression analysis of IRX3 in TRAMP tumors compared to normal prostate. Filled bars are those that were shown to be hypermethylated by RLGS analysis. A Irx3 NotI Site N LPD11 LPD1 LPD13 LPD8 B Irx3 mrna Expression N4 N1 N2 N3 LPD11 LPD14 LPD12 LPD13 LPD15 LPD2 LPD6 LPD8 LPD4 LPD5 LPD3 LPD1 LPD9 LPD7 LPD10 15

16 Figure 2. TRAMP tumors displaying downstream hypermethylation of the CDKN2A locus show increased mrna expression of p19 and p16. A-B) p19 and p16 mrna expression in LPD, MET and AIP tumors grouped by either methylated or unmethylated RLGS status. no AIP samples are unmethylated at this locus by RLGS analysis C-D) p19 and p16 mrna expression in N, PIN, WD, EPD, and LPD samples. Mann-Whitney test p-values: ** p < 0.005; * p < 0.01, for each group compared to normal prostate. A p19 Normalized Expression LPD Meth LPD Unmeth MET Meth MET Unmeth RLGS Status AIP Meth AIP Unmeth B p16 Normalized Expression LPD Meth LPD Unmeth MET Meth MET Unmeth RLGS Status AIP Meth AIP Unmeth C p19 Copy # / 18s Copy # mrna Expression ** ** ** ** N PIN WD EPD LPD D p16 Copy # / 18s Copy # mrna Expression ** ** ** * N PIN WD EPD LPD 16

17 Figure 3. p19 and p16 expression are decreased in TRAMP C2D cell line with DAC treatment. A) p19 and p16 mrna expression in TRAMP C2D cell line without and with DAC treatment for 48 hours. B) Bisulfite pyrosequencing analysis of the hypermethylated CDKN2A downstream region without and with DAC treatment in TRAMP C2D cell line. A Copy # / 18s Copy # 10 0 mrna Expression 10 2 C2 C2 C B 10-1 p19 p16 C2D Control C2D 0.5μM DAC C2D 1μM DAC 17

18 Figure 4. Bisulfite sequencing analysis of CDKN2A, Cacna1a, GSC and NRXN2 loci. A) Upper: diagram of the Cdkn2a locus. Open rectangles, p19 exons; hashed rectangles, p16 exons; lines, introns; right arrows, transcriptional start sites; vertical arrow, position of the NotI site identified by RLGS; black rectangles, CpG islands; horizontal line with circles (A-C), regions analyzed by sodium bisulfite sequencing. Lower: sodium bisulfite sequencing data of regions A-C in normal prostate and TRAMP samples. Percent methylation (averaged over the entire sequenced region), from at least ten sequenced clones per sample, is plotted. B) Upper: diagram of the Cacna1a locus. Right arrow, transcriptional start site; open rectangles, exons; lines, introns; vertical arrow, position of the NotI site identified by RLGS; black bar, CpG island; horizontal line with circles (A and B), regions analyzed by sodium bisulfite sequencing. Lower: sodium bisulfite sequencing data of regions A and B in normal prostate and TRAMP samples. Percent methylation (averaged over the sequenced entire region), from at least ten sequenced clones per sample, is plotted. C-D) MAQMA bisulfite sequencing results for the Gsc and Nrxn2 loci. Percent methylation (averaged over the sequenced entire region), plotted against sample type and RLGS methylation status. A p19 ARF p16 INK4a Not I Site B Cacna1a NotI Site 1 β 1 α Region A B C % Methylation Kb Kb Kb Kb 100 N N 2 PIN 1 PIN 2 WD 1 40 WD 2 EPD EPD 2 LPD 1 A B C LPD 2 Bisulfite Sequenced Region Region A B Kb Kb 80 N 1 N 2 % Methylation 60 PIN A B Bisulfite Sequenced Region PIN 2 WD 1 WD 2 EPD 1 EPD 2 EPD 3 LPD 1 LPD 2 C GSC D NRXN2 LPD LPD MET MET AIP AIP Meth Unmeth Meth Unmeth Meth Unmeth RLGS Status LPD LPD MET MET AIP AIP Meth Unmeth Meth Unmeth Meth Unmeth RLGS Status 18

19 Table 3. Inheritance analysis of Dnmt1 hypomorphic alleles Genotype WT N/+ R/+ N/R # Mice analyzed Mendelian Ratio Figure 5. Animal, urogenital (UG), and prostate weight in Dnmt1 hypomorphic mice. A) Weight of animal at sacrifice in Dnmt1 hypomorphic mice in all four genotypes (WT, N/+, R/+, N/R) at either 15 or 24 weeks of age. B) Urogenital weight at sacrifice in Dnmt1 hypomorphic mice in all four genotypes (WT, N/+, R/+, N/R) at either 15 or 24 weeks of age. C) Prostate weight at sacrifice in Dnmt1 hypomorphic mice in all four genotypes (WT, N/+, R/+, N/R) at either 15 or 24 weeks of age. Mann-Whitney test p- values: ** p < 0.005; * p < 0.01, for each group compared to WT for each time point. A Animal Weight (Gms) Dnmt1 Hypomorphic Mice B Dnmt1 Hypomorphic Mice ** ** 0.8 * *** *** UG Weight (Gms) C Prostate Weight (Gms) WT - 15 weeks N/ weeks R/ weeks N/R - 15 weeks WT - 24 weeks N/ weeks Dnmt1 Hypomorphic Mice ** WT - 15 weeks N/ weeks R/ weeks N/R - 15 weeks WT - 24 weeks N/ weeks R/ weeks N/R - 24 weeks R/ weeks N/R - 24 weeks 0.0 WT - 15 weeks N/ weeks R/ weeks N/R - 15 weeks WT - 24 weeks N/ weeks R/ weeks N/R - 24 weeks 19

20 Figure 6. Global Methylation is decreased in Dnmt1 hypomorphic mice in both prostate and liver. A) Global methylation levels in prostate measured as %5mdC by liquid chromatography mass spectrometry in Dnmt1 hypomorphic mice in all four genotypes (WT, N/+, R/+, N/R) at either 15 or 24 weeks of age. B) Global methylation levels in liver measured as %5mdC by liquid chromatography mass spectrometry in Dnmt1 hypomorphic mice in all four genotypes (WT, N/+, R/+, N/R) at either 15 or 24 weeks of age. Mann-Whitney test p-value: * p < 0.01, for each group compared to WT for each time point. A Global Methylation in Prostate [% 5mdC/dG] * B [% 5mdC/dG] Global Methylation in Liver 0 WT - 15 weeks N/ weeks R/ weeks N/R - 15 weeks WT - 24 weeks N/ weeks R/ weeks N/R - 24 weeks 0 WT - 15 weeks N/ weeks R/ weeks N/R - 15 weeks WT - 24 weeks N/ weeks R/ weeks N/R - 24 weeks 20

21 Figure 7. Animal, urogenital, and prostate weight in Dnmt1 Hypomorphic TRAMP mice. A) Weight of animal at sacrifice in Dnmt1 hypomorphic TRAMP mice in all three genotypes (WT, N/+, R/+) at either 15 or 24 weeks of age. B) Urogenital weight at sacrifice in Dnmt1 hypomorphic TRAMP mice in all three genotypes (WT, N/+, R/+) at either 15 or 24 weeks of age. C) Prostate weight at sacrifice in Dnmt1 hypomorphic TRAMP mice in all three genotypes (WT, N/+, R/+) at either 15 or 24 weeks of age. Mann-Whitney test p-value: * p < 0.05, for each group compared to WT for each time point. A Animal Weight (Gms) Dnmt1 Hypomorph TRAMP Mice 40 * WT - 15 weeks N/ weeks R/ weeks WT - 24 weeks N/ weeks R/ weeks B UG Weight (Gms) Dnmt1 Hypomorph TRAMP Mice * WT - 15 weeks N/ weeks R/ weeks WT - 24 weeks N/ weeks R/ weeks C Prostate Weight (Gms) Dnmt1 Hypomorph TRAMP Mice 15 * WT - 15 weeks N/ weeks R/ weeks WT - 24 weeks N/ weeks R/ weeks 21

22 Publication 1: Stage-specific alterations of Dnmt expression, DNA hypermethylation, and DNA hypomethylation during prostate cancer progression in the TRAMP model Molecular Cancer Research (In press) Shannon R. Morey Kinney 1, Dominic J. Smiraglia 2, Smitha R. James 1, Michael T. Moser 1, Barbara A. Foster 1, and Adam R. Karpf 1 Departments of Pharmacology and Therapeutics 1 and Cancer Genetics 2, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY Send Reprint Requests to: Dr. Adam R. Karpf, Department of Pharmacology & Therapeutics, Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY Phone: , adam.karpf@roswellpark.org Running title - Altered DNA methylation during TRAMP progression Keywords - methyltransferase TRAMP, DNA methylation, epigenetics, prostate cancer, DNA Grant Information: NIH R21CA (ARK), NIH 5T32CA (SRMK), DOD PC (SRMK), NCI Center Grant CA16056 (Roswell Park Cancer Institute). Abstract We analyzed DNA methyltransferase protein expression and DNA methylation patterns during four progressive stages of prostate cancer in the Transgenic Adenocarcinoma of Mouse Prostate (TRAMP) model, including prostatic intraepithelial neoplasia (PIN), well differentiated tumors (WD), early poorly differentiated tumors (EPD), and late poorly differentiated tumors (LPD). Dnmt1, Dnmt3a, and Dnmt3b protein expression are increased in all stages, coinciding with overexpression of E2F gene targets, linking this alteration to Rb inactivation by Large T antigen. After normalization to Cyclin A to account for cell cycle regulation, Dnmt proteins remained over-expressed in all stages except LPD. Restriction Landmark Genomic Scanning (RLGS) analysis of locus-specific methylation revealed a high incidence of hypermethylation only in poorly differentiated (EPD and LPD) tumors. Several genes identified by RLGS showed hypermethylation of downstream regions correlating with mrna overexpression, including p16ink4a, p19arf, and Cacna1a. Parallel gene expression and DNA methylation analyses suggests that gene overexpression precedes downstream hypermethylation during prostate tumor progression. In contrast to gene hypermethylation, genomic DNA hypomethylation, including hypomethylation of repetitive elements and loss of genomic 5mdC, occurred in both early and late stages of prostate cancer. Statistical correlation analyses reveal that locus-specific hypermethylation and global hypomethylation are not associated in TRAMP. Notably, Dnmt1 and Dnmt3b over-expression correlated with global DNA hypomethylation, but 22

23 not locus-specific hypermethylation, suggesting the existence of a regulatory loop responsive to global DNA hypomethylation that involves specific Dnmts. In summary, our data reveal the temporal relationship between key alterations of the DNA methylation pathway occurring during prostate tumor progression. Introduction DNA methylation is deregulated in cancer such that the promoter regions of tumor suppressor genes become hypermethylated, resulting in gene silencing, while, on a global level, DNA becomes hypomethylated, potentially leading to genomic instability (4, 5). In human prostate cancer, both of these mechanisms have been observed (6). In addition, deregulated expression of DNA methyltransferase (Dnmt) proteins is seen in human prostate cancer. These data provide compelling circumstantial evidence of a role for these alterations in prostate cancer development. However, it is difficult to assess the functional contribution of these alterations to prostate cancer development using only human clinical samples. Moreover, the relative timing of and relationship between distinct DNA methylation pathway alterations during prostate tumor progression has not been assessed in an experimentally tractable model system. To this end, we and others have recently established TRAMP (Transgenic Adenocarcinoma of Mouse Prostate) as a suitable mouse model to investigate the role of altered DNA methylation in prostate cancer development (2, 7, 8). We have shown that late stage primary tumors and metastases from TRAMP mice display increased Dnmt expression, locus-specific nonrandom CpG island hypermethylation, and hypomethylation of repetitive DNA elements (2). In addition, others have demonstrated, using pharmacological inhibition of Dnmt enzymes, that DNA hypermethylation contributes to the development of primary cancer in both intact and castrated TRAMP mice (7, 8). Taken together, these data suggest that the TRAMP model may be particularly useful to clarify the role of DNA methylation pathway alterations in prostate cancer development. One notable finding of our previous study was that TRAMP tumors frequently display overexpression of p19arf (p19) and p16ink4a (p16), correlating with hypermethylation of a shared downstream region (exon 3) of the Cdkn2a locus (2). The relevance of this event to human prostate cancer is supported by the prior observation that p16 gene up-regulation and downstream hypermethylation also occur in human prostate cancer. Using Restriction Landmark Genomic Scanning (RLGS), we identified several other genes that were hypermethylated in downstream regions in TRAMP, relative to normal prostate, suggesting that this phenomenon may be widespread (2). Previous work in other systems has also reported hypermethylation of actively transcribed downstream gene regions in cancer. However, it remains unclear whether gene overexpression in cancer occurs prior or subsequent to downstream DNA hypermethylation. In the current study we sought to define the relationship between disease stage, Dnmt expression, DNA hypermethylation, and DNA hypomethylation in prostate cancer. For this purpose, we selected TRAMP prostate samples from four distinct groups (prostatic intraepithelial neoplasia (PIN), well-differentiated tumors (WD), early poorly differentiated tumors (EPD), and late poorly differentiated tumors (LPD)) for analysis, for comparison to non-transgenic strain matched normal mouse prostates. In each sample set we measured Dnmt1, Dnmt3a, and Dnmt3b protein expression by Western blot, locus-specific methylation using RLGS, and global methylation using Liquid 23

24 Chromatography-Mass Spectrometry (LC-MS) detection of 5-methyldeoxycytidine (5mdC), and bisulfite pyrosequencing of the B1 repetitive element. In addition, we examined the relationship between gene overexpression and downstream hypermethylation in TRAMP, via comparative mrna expression and DNA methylation analysis of p16ink4a, p19arf, and Cacna1a in staged tumor samples. Importantly, we performed statistical correlation analyses to assess the relationship between each of these parameters during tumor progression. Our findings reveal key aspects of the relationship between distinct alterations of the DNA methylation pathway occurring during prostate tumor progression. Results Multi-stage Prostate Cancer (CaP) Progression in TRAMP. In this study, we utilized prostate tumors from TRAMP mice, as well as normal prostates from nontransgenic, strain-matched mice (Fig. 1A). We grouped TRAMP samples based on differentiation status, age, and prostate weight into the following four categories: Prostatic Intraepithelial Neoplasia (PIN, weeks, gm, n = 35), welldifferentiated tumors (WD, weeks, gm, n = 25), early poorlydifferentiated tumors (EPD, weeks, gm, n = 12), and late poorlydifferentiated tumors (LPD, weeks, gm, n = 12) (Fig. 1A). This grouping is based on previous studies showing that age and prostate weight directly correlate with tumor progression in TRAMP (9). PIN samples are normal in weight, but microscopically display neoplasia and hyperplastic infolding of the epithelial layer into the luminal space of the gland (Fig. 1, A and B). WD samples are larger than normal prostates, but were not palpable at necropsy. The majority of the disease in these samples is well differentiated glandular epithelium (Fig. 1B). EPD samples are from the same age range as WD samples (15-20 weeks), but were palpable at necropsy and histologically demonstrated predominantly sheets of poorly differentiated epithelial cells (Fig. 1, A and B). LPD tumors, from week old mice, were very large and show poorly differentiated late stage disease (Fig. 1, A and B). Hematoxylin and eosin (H&E) staining was used to stage a subset of samples and confirmed the assigned groupings (Fig. 1B and data not shown). Dnmt protein expression during multi-stage CaP progression. We initially examined Dnmt1, Dnmt3a, and Dnmt3b protein expression in normal prostates and the four sets of TRAMP samples described above using Western blot analysis. Dnmt1 expression is significantly elevated in PIN and WD and its level increases further in late stage (EPD and LPD) samples (Fig. 2A and B). Dnmt3a and Dnmt3b also show elevated expression in PIN and WD, which increases in EPD and LPD tumors (Fig. 2A, C-D). We measured Cyclin A, to normalize Dnmt expression, as Dnmt expression is cell cycle regulated with high level expression restricted to S phase. Cyclin A expression is robustly increased only in late stage (EPD and LPD) disease (Fig. 2A and E). As expected based on our previous work, there was a strong association between the expression of each Dnmt and the expression of Cyclin A, SV40 Large T antigen and E2F1 ( and data not shown). Notably, after normalization to Cyclin A, Dnmt1 and Dnmt3b expression are significantly upregulated in PIN, WD, and EPD, but no longer in LPD tumors (Fig. 2F and H). Dnmt3a is upregulated only in PIN and WD tumors after normalization to Cyclin A (Fig. 2G). Taken together, these data indicate that increased 24

25 Dnmt protein expression is not solely accounted for by increased cell proliferation, and may be most biologically significant at early progression stages in TRAMP. Locus-specific DNA hypermethylation during multi-stage CaP progression. We next utilized RLGS to examine global CpG island methylation patterns in TRAMP samples of each progression stage. RLGS is a two-dimensional gel analysis of radiolabeled, methylation sensitive enzyme-restricted DNA fragments. When comparing RLGS gel patterns, spot loss and spot gain correspond to DNA hypermethylation and DNA hypomethylation events, respectively. RLGS allowed for the identification of hypermethylation events in TRAMP which, in the vast majority of instances, were confined to late stage (EPD or LPD) disease (examples shown in Fig. 3A and B). A low level of both hypermethylation and hypomethylation events were observed in PIN and WD samples, while EPD and LPD tumors showed a substantial increase in hypermethylation events (Fig. 3C and D). In addition, the number of hypermethylated loci from tumor to tumor was variable within the EPD, and particularly the LPD, groups (Fig. 3D). We identified the genes corresponding to different RLGS spots using cloning techniques described previously (Table 1). A number of these loci were hypermethylated at high frequency in EPD and LPD (Table 1), suggesting that methylation of these loci are under positive selection during prostate cancer progression in TRAMP. Downstream hypermethylation and increased gene expression. We previously reported that overexpression of p19 and p16 correlated with the downstream hypermethylation at the shared exon 3 of the Cdkn2a locus in late stage TRAMP tumors (2). Several other genes also display hypermethylation in downstream regions in TRAMP tumors, providing further evidence of the potential importance of this phenomenon (Table 1, genes in bold). The staged progression model we describe here allows for an investigation of the relative timing of gene overexpression and downstream hypermethylation. We find that p19 and p16 are over-expressed in all stages analyzed, as compared to normal prostate, indicating that overexpression is an early event (Fig. 4A and B). In contrast, RLGS indicated that hypermethylation of the NotI site at exon 3 of the Cdkn2a locus was exclusively found in late stage (EPD and LPD) samples (Table 1). Bisulfite sequencing confirmed that TRAMP tumors sometimes fail to show Cdkn2a exon 3 hypermethylation, despite the fact that overexpression is uniformly observed in these lesions (Fig. 4C). These data suggest that gene overexpression precedes downstream hypermethylation at the Cdkn2a locus during tumor progression. Moreover, downstream hypermethylation at the Cdkn2a locus in TRAMP is rarely accompanied by hypermethylation at the p19 or p16 promoter regions (Fig. 4C). To investigate this phenomenon at a distinct locus, we measured the expression and methylation of the calcium channel gene Cacna1a, which is frequently methylated in a 3 region (exon 33) in TRAMP (Table 1). Cacna1a is overexpressed only in late stage (EPD and LPD) tumors, paralleling its exclusive methylation in these stages (Fig. 5A, Table 1). Notably, overexpression occurred in all analyzed late stage (EPD and LPD) samples, while downstream hypermethylation occurred only in approximately half of these samples (Fig. 5A, Table 1). Bisulfite sequencing further demonstrated that this downstream region of Cacna1a, but not its promoter region, is methylated in TRAMP (Fig. 5B). Taken together, these data suggest that, similar to Cdkn2a genes, overexpression of Cacna1a precedes its downstream hypermethylation. Interestingly, a low but significant level of methylation at the Cacna1a locus was seen in both normal 25

26 prostates and early stage samples (Fig. 5B). This situation may be analogous to certain genes that are partially methylated in normal human prostate and become hypermethylated in human prostate cancer. DNA hypomethylation during multi-stage CaP progression. In addition to gene specific DNA hypermethylation, global DNA hypomethylation appears to contribute to oncogenesis. In TRAMP, we previously found increased variability but no consistent changes in 5mdC levels in late stage TRAMP tumors and metastases as compared to normal strain-matched prostates (2). We hypothesized that global hypomethylation may be an early event during TRAMP tumor development that could have been missed in our previous study. To test this hypothesis, we measured 5mdC levels by LC-MS as well as the methylation level of the common murine repetitive element B1 using quantitative bisulfite pyrosequencing, in the four stages of TRAMP samples described earlier (Fig 1A). Notably, 5mdC levels were significantly decreased in WD and EPD tumors (Fig. 6A). At the latest stage, LPD, this effect was lost; however increasing variability from tumor to tumor was apparent (Fig 6A). In contrast to 5mdC levels, the B1 repetitive element is significantly hypomethylated in all four progression stages measured, but more dramatically in the later stages (Fig. 6B). Analyzed over the entire data set, 5mdC levels directly correlated with B1 methylation (Spearman Rank Correlation r = 0.30, p=0.04). These experiments demonstrate that genomic DNA hypomethylation occurs as an early event during prostate tumorigenesis in TRAMP, and persists and/or increases in advanced stages. Relationship between DNA methylation pathway alterations in TRAMP. We took advantage of our unique data set to examine the relationship between Dnmt protein expression, DNA hypermethylation, and DNA hypomethylation during prostate tumorigenesis. To examine the potential link between DNA hypermethylation and DNA hypomethylation, we compared the extent of RLGS spot loss to 5mdC levels or B1 element methylation status in all samples (Fig. 6C and D). Notably, there was no association between DNA hypermethylation and either parameter of global DNA hypomethylation, suggesting that hyper- and hypomethylation are independently controlled in TRAMP. The lack of association was still seen when only late stage (EPD and LPD) samples, which show a much higher incidence of DNA hypermethylation (Fig 3), were analyzed (Fig. 6E and F). We also compared Dnmt1, Dnmt3a, and Dnmt3b protein expression, after normalization to Cyclin A, to DNA hypermethylation and DNA hypomethylation. Dnmt protein expression did not correlate with RLGS spot loss (DNA hypermethylation) in all TRAMP samples, suggesting that locus-specific DNA hypermethylation may result from a defect in methylation targeting rather than from altered Dnmt expression (Table 2). Again, a lack of association between these parameters was maintained when only late stage tumors (EPD and LPD) were analyzed (data not shown). Interestingly, expression of Dnmt1 and Dnmt3b, but not Dnmt3a, was inversely correlated with 5mdC levels (Table 2). For B1 methylation, the same trend was apparent, but did not reach statistical significance (Table 2). This intriguing finding suggests that Dnmt1 and Dnmt3b protein overexpression in prostate cancer may reflect a regulatory loop responsive to global DNA hypomethylation. 26